Role of α-Asp181, β-Asp192, and γ-Asp190 in the distinctive subunits of human NAD-specific isocitrate dehydrogenase

Human NAD-dependent isocitrate dehydrogenase (IDH) is allosterically activated by ADP by lowering the K-m for isocitrate. The enzyme has three subunit types with distinguishable sequences present in the approximate ratio 2 alpha:1 beta:1 gamma and, per tetramer, binds 2 mol of each ligand. To evaluate whether the subunits also have distinct functions, we replaced equivalent aspartates, one subunit at a time, by asparagines; each expressed, purified enzyme was composed of one mutant and two wild-type subunits. The aspartates were chosen because beta-Asp(192) and gamma-Asp(190) had previously been affinity labeled by a reactive ADP analogue and alpha-Asp(181) is equivalent based on sequence alignments. The alpha-D181N IDH mutant exhibits a 2000-fold decrease in V-max, with increases of 15-fold in the K(m)s for Mn(II) and NAD and a much smaller change in the K-m for isocitrate. In contrast, the V-max values of the beta-D192N and gamma-D190N IDHs are only reduced 4-5-fold as compared to wild-type enzyme. The K-m for NAD of the beta-D192N enzyme is 9 times that of the normal enzyme with little or no effect on the affinity for Mn(II) or isocitrate, while the K(m)s for coenzyme and for Mn(II) of the gamma-D190N enzyme are 19 and 72 times, respectively, that of the normal enzyme with a much smaller effect on the K-m for isocitrate. Finally, all three mutant enzymes fail to respond to ADP by lowering the K-m for isocitrate, although they do bind ADP. Thus, these aspartates are close to but not in the ADP site and are required for communication between the ADP and isocitrate sites. These results demonstrate that alpha-Asp(181) is the only one of these aspartates essential for catalysis. beta-Asp(192) is a determinant of the enzyme's affinity for NAD, as is gamma-Asp(190), while gamma-Asp(190) also influences the enzyme's affinity for metal ion. We conclude that the NAD and ADP sites are shared between alpha- and beta- and alpha- and gamma-subunits, and the Mn(II) site is shared between alpha- and gamma-subunits, while the alpha-subunit is essential for catalysis. Although alpha-Asp(181), beta-Asp(192), and gamma-Asp(190) may have derived from a common progenitor, these aspartates of the three subunits have evolved distinct functions.